Interactions of Catalytic Enzymes with n-Type Polymers for High-Performance Metabolite Sensors

The tight regulation of the glucose concentration in the body is crucial for balanced physiological function. We developed an electrochemical transistor comprising an n-type conjugated polymer film in contact with a catalytic enzyme for sensitive and selective glucose detection in bodily fluids. Despite the promise of these sensors, the property of the polymer that led to such high performance has remained unknown, with charge transport being the only characteristic under focus. Here, we studied the impact of the polymer chemical structure on film surface properties and enzyme adsorption behavior using a combination of physiochemical characterization methods and correlated our findings with the resulting sensor performance. We developed five n-type polymers bearing the same backbone with side chains differing in polarity and charge. We found that the nature of the side chains modulated the film surface properties, dictating the extent of interactions between the enzyme and the polymer film. Quartz crystal microbalance with dissipation monitoring studies showed that hydrophobic surfaces retained more enzymes in a densely packed arrangement, while hydrophilic surfaces captured fewer enzymes in a flattened conformation. X-ray photoelectron spectroscopy analysis of the surfaces revealed strong interactions of the enzyme with the glycolated side chains of the polymers, which improved for linear side chains compared to those for branched ones. We probed the alterations in the enzyme structure upon adsorption using circular dichroism, which suggested protein denaturation on hydrophobic surfaces. Our study concludes that a negatively charged, smooth, and hydrophilic film surface provides the best environment for enzyme adsorption with desired mass and conformation, maximizing the sensor performance. This knowledge will guide synthetic work aiming to establish close interactions between proteins and electronic materials, which is crucial for developing high-performance enzymatic metabolite biosensors and biocatalytic charge-conversion devices.


Synthesis of the polymers
All reactions were performed in oven-dried glassware under a nitrogen atmosphere using standard Schlenk techniques unless otherwise stated. Monomer (3,3'-bis(2-(2-(2-methoxy ethoxy)ethoxy)ethoxy)-[2,2'-bithiophene]-5,5'-diyl)bis(trimethyl stannane) was synthesized as previously reported. 1 All commercially available materials were used as received. 1 4 . The solvent was removed in vacuo, and the solid residue was washed with MeOH and hot acetone to give the title product as an orange solid (0.14 g, 24%). 1 Figure S2. 13     in anhydrous DMF (4 mL), and the resulting mixture was heated to 120 °C for 16 h. The reaction mixture was allowed to reach room temperature, and the solvent was removed under a vacuum.
The green residue was suspended in acetone, filtered, and thoroughly washed with hot acetone and chloroform to give the title product as a green solid with a yield of 64% (12.      The arrows in the output curves indicate the increase in the gate voltage. P-ZI could not be operated using this configuration. Figure S7. O 2 sensitivity of n-type films. The current response of n-type polymer-coated Au electrodes to O 2 was evaluated using a three-electrode setup. The working electrode was the GOx adsorbed n-type film, the reference electrode was Ag/AgCl, and the counter electrode was a Pt coil. The electrode was first immersed in a de-gassed electrolyte in a glovebox under an N 2saturated atmosphere. Once a stabilized current was achieved, the glovebox door was opened (represented by the arrow) to allow the ambient O 2 to enter and dissolve in the electrolyte.
For P-90 and P-100, the dip in current corresponds to the pipetting of fresh electrolyte into the system. For P-100B, two different films were used (one measured in N 2 , the other in ambient) as the film was delaminating when biased for an extended period.     frequency (f n ) and the right one of the dissipation (d n ) for harmonics 5 th , 7 th , and 9 th (n represents the harmonic number) are shown. After the films were fully swollen in PBS (no change in QCM-D signals), GOx was introduced to the chamber ("+ Gox"). After an hour of adsorption, the films were rinsed with PBS to remove any unbound species ("Rinse").   Δf plots (7 th harmonic). The numbers define the linear regions in the plots with different slopes.
The red dot demarks the transition from the adsorption process to the rinsing process.    Table S2. The deconvolution of the N 1s XPS spectra is shown in Figure 6. Non-polar, aliphatic residues 7.6 Figure S20. High-resolution of C 1s XPS spectra of polymer films. Table S4. Deconvolution of the XPS spectra is shown in Figure S20.    Table S6. Deconvolution of the C 1s XPS spectra is shown in Figure S21.  We observe other deconvoluted peaks in the S 2p spectra after GOx adsorption (e.g., methionine -MET, and possibly cysteine -CYS, [amino acids]). In particular, we observe a peak at approximately 165.5-165.8 eV, which is more prominent in P-100 than in the other polymers.

Bonds of interest
Many chemical groups can lead to this peak, including cysteine amino acid, 5 and oxidized methionine sulfur 6 since this residue is more exposed to the outer environment (i.e. reactive oxygen species) due to the hypothesized expanded conformation of GOx on P-100. Nevertheless, given that P-100 generally retains the least amount of enzyme on its surface, stronger XPS signals suggest a higher concentration of the investigated species, which is only possible if more of this species is exposed to the XPS beam. Table S7. Deconvolution of the S 2p XPS spectra is shown in Figure S22.   Table S8. Binding energy shifts of the C 1s peaks after enzyme adsorption. The C 1s spectra before and after enzyme adsorption are displayed in Figures S20 and S21 whereas very low ellipticity above 210 nm and negative bands near 195 nm correspond to disordered conformation. 10 Typically, the difference in absorption between right and left-polarized light in the far-and near-UV regions (190 to 250 and 250 to 300 nm, respectively) is recorded as a fingerprint of the biomolecule structure.

Supplementary Discussion 1. Effect of surface charge and wettability on protein adsorption.
Proteins are thought to undergo translational and rotational motions to find a preferred orientation before adsorption, dictated by the properties of the underlying substrate surface. 11 The favored orientation of a protein on the surface is related to its free energy minimum, which depends on various surface-related parameters, such as Coulomb and Van der Waals interactions, hydrogen bonds, and entropy gain of solvent molecules or counter-ion release. 12 The substrate-protein interactions may lead to an increase in protein's free energy, ultimately maximizing its footprint through conformational reorganization. 12 11 The authors demonstrated that GOx adsorbed in different configurations depending on the underlying surface charge (Figures 3 and S9). On positively charged surfaces, such as P-100B, the negatively charged GOx (isoelectric point -PI 4.2) can adsorb in a "standing" fashion, where the substrate-binding domain is accessible to glucose. However, as the surface charge density decreases, Van der Waals interactions become more prominent, and more possible orientations can occur where the protein ultimately preferentially adopts an unfavorable "frontlying" position where the substrate-binding domain is inaccessible, as assumed to be the case for P-ZI. On negatively charged surfaces (P-75, P-90, and P-100), although GOx is also overall negatively charged, it can still adsorb through its positively charged lysine or neutral residues (Figures S9 and Table S3), adopting a "back-lying orientation" with the substrate-binding site facing outward and the redox-active center facing the polymer surface. 11 In addition, the relatively high ionic strength of the medium (1X PBS) is expected to influence GOx adsorption due to charge screening by ions in the solution. In some cases, the screening effect from the electrolyte has been shown to weaken electrostatic interactions, leaving only Van der Waals interactions at play. 12 However, the screening effect is not expected to be beneficial to the adsorption of GOx on a negatively charged surface. This effect may contribute to the decrease in the adsorbed mass for the mixed alkyl/glycol series, where the least negatively charged surface has the highest adsorbed mass. For the zwitterionic surface (P-ZI), only the Van der Waals interactions are expected to occur, suggesting an unfavorable front-lying orientation of GOx on the polymer surface.
Effect of surface wettability. Protein adsorption is less favored on hydrophilic surfaces due to the energetic cost of surface dehydration, where a contact angle of 65° has been reported as the hydrophilicity threshold for adsorption. 14 However, the literature is inconsistent on this matter. 12,14 For example, Anand et al. postulated that changing surface polarity affects the adsorbed protein amount (increasing as the surface becomes less polar) and that increased surface polarity destabilizes the proteins (unfolding) due to stronger protein-protein and protein-surface interactions. 15 The authors suggested that a less polar surface leads to a more rigid adsorbed layer.
In the case of glycoproteins, such as GOx, the hydrophobic side chains are directed inward. 16 Protein adsorption on hydrophobic/apolar surfaces is expected to lead to substantial conformational changes. 16

Supplementary Discussion 2. Δd vs. Δf analysis.
We investigate the viscoelastic properties of the GOx adsorbed layer by plotting Δd as a function of Δf (Figures 4d, S16, and Table S1). The Δd vs. Δf plots represent viscoelastic changes in the protein layers as a function of the adsorbed mass. 17 Three main factors generally accounted for changes in dissipation: dissipations located at the (i) protein-substrate interface and (ii) proteinliquid interface (including effects of a change in surface roughness), and (iii) within the protein layer (including effects of trapped liquid as the available space changes dynamically with the adsorption process). 17 The Δd vs. Δf plots contain a series of linear regions with their characteristic slopes, where a high value indicates the formation of a loose protein layer. 17 Figure S16 shows that all polymers, except P-ZI, presented 3 linear regions in the adsorption process. Phase  indicates a relatively rigid (low dissipation vs. frequency ) attachment until a critical surface coverage is reached. Phase  exhibits either the attachment of a viscoelastic stratum onto the first layer or loose (i.e., imperfectly coupled) binding of an additional rigid layer onto the first. The adsorption of this layer seems to be partially reversible, as illustrated in the rinsing process. Phase  displays a further increase in the frequency, with a slight increase in dissipation, related to film thickening and possibly the removal of water molecules between loosely bound adlayers. Increasing the EG content decreases the rigidity of the first layer adsorbed on the films. P-ZI presents a unique case, revealing a two-step adsorption process. In the P-ZI phase  stabilization, we observe a slight decrease in the d value with an increase in f, indicating a thickening of the enzyme layer and the possible removal of water molecules from the loosely bound adlayers.
During the washing phase, a rapid decrease in d was observed for all polymers, concomitant with a slight f decrease, which most likely is due to the outer (i.e., film-solvent located) loosely adsorbed GOx molecules being removed by the buffer. Afterward, during the stabilization of the rinsing process, we observe the opposite behavior for P-75/P-90 and P-100/P-100B. P-75 and P-90 display a relatively constant d value (or slightly decreasing) with a decrease in the f value, suggesting the formation of a looser protein layer rather than a more rigid one. In contrast, P-100 and P-100B exhibit a slight increase in f and d values (more evident for P-100B) after stabilization, suggesting a configuration change in the existing bound layers, possibly the formation of a looser and more hydrated structure upon introducing a new buffer (especially for P-100B). On the other hand, the washing phase of P-ZI films shows a sharp decrease in d with a slight decrease in the f value, suggesting a significant stiffening of the layer. This is also indicated by the "negative" value of the dissipation upon rinsing (negative since we take the swollen polymer as the baseline). Upon stabilization following the washing step, an increase in d occurred with a relatively constant f, indicating a possible relaxation of the enzyme layer.